Malignant tumors often demonstrate a resistance to radiation therapy and chemotherapy. The resistance of physiologically hypoxic regions within solid tumors during cancer treatment is an important reason for the failure of radiotherapy and chemotherapy to eradicate such tumors.
Investigators have made progress in understanding the basic cellular and molecular mechanisms of therapeutic resistance. Many of these investigative efforts have focused on intrinsic cellular characteristics. However, there is an aspect of therapeutic resistance which is related to the physiologic and biochemical state of the cell, and not to intrinsic cellular properties. In other words, cells which are otherwise sensitive to cytotoxin treatment under normal physiologic conditions, are resistant because of their particular physiologic state within the tumor.
For example, it has long been known that hypoxic, i.e., oxygen deficient, cells are relatively resistant to killing by radiation. To achieve the same proportion of cell kill, about three times the radiation dose is required for hypoxic cells compared to the radiation dose required for well-oxygenated cells. Thus oxygen has the ability to sensitize cells to ionizing radiation at clinical radiation doses. Overcoming this resistance of hypoxic cells has been investigated as a means of improving the efficacy of ionizing radiation.
Multiple mechanisms have been proposed to explain hypoxic resistance to radiation therapy and chemotherapy. The proposed mechanisms involve kinetic, metabolic, and physical factors. For example, hypoxic cells frequently are noncycling and therefore are refractory to proliferation-dependent cytotoxic drugs. In addition, the cell can be in a metabolically-compromised state and unable to concentrate and activate potentially-effective agents. The distance between a cell and blood vessels also can be greater than the diffusion distance of many chemotherapeutic agents.
Efforts to overcome hypoxia in clinical cancer treatments have involved the development of hypoxic cell radiosensitizers and chemosensitizers which substitute for, or mimic, oxygen.
Cell kill by ionizing radiation is caused by damage to the DNA. The target radical on the DNA, designated "DNA-", is produced by either direct ionization or reaction with hydroxyl radicals produced radiolysis of neighboring water molecules. Reaction with oxygen produces a peroxyl radical, DNA-O.sub.2., which forms products leading to irreversible DNA damage. Radiosensitizers are designed to mimic oxygen by reacting with DNA radicals to form covalent adducts at the radical sites.
As discussed hereafter, the ability of oxygen or a sensitizer to enhance cell kill is reflected in the enhancement ratio, i.e., OER for oxygen and SER (sensitizer enhancement ratio) for the sensitizer. The OER is dependent on the concentration of oxygen present at the target at the time of irradiation. Similarly, the oxygen-mimicking effect of a hypoxic cell sensitizer (SER) depends primarily on the concentration of sensitizer at the target at the time of irradiation. However, the oxygen-mimicking sensitizers preferentially affect hypoxic cells, which typically comprise only about 20% of the tumor. Therefore, in estimating the degree of enhancement of cell kill, the SER applies only to hypoxic cells, not to the entire tumor.
It is well established that bioreductive compounds, such as nitroimidazole-based compounds, potentiate the cytotoxic effects of radiation and several chemotherapeutic agents towards hypoxic tumor cells, both in vitro and in vivo. Bioreductive compounds are readily activated by metabolic reduction in a hypoxic environment and enhance the susceptibility of hypoxic tumor cells to radiation and conventional anticancer drugs.
Bioreductive agents typically are compounds of high electron affinity. Bioreductive agents have the ability to kill hypoxic cells directly because of their preferential reductive metabolism under hypoxic conditions, where the limited oxygen concentration cannot significantly antagonize the reduction process. In addition, solid tumors develop physiological hypoxia to a greater degree than normal tissues, and evidence exists that tumor cells have relatively high levels of reductive enzymes.
Bioreductive agents in hypoxic cells therefore mimic the oxygen effect in oxygenated cells during irradiation, and cause fixation of radiation-induced damage to DNA or other vital macromolecules. Thus, bioreductive agents act as radiosensitizers of hypoxic cells. Bioreductive agents can also act as chemosensitizers for conventional anticancer drugs by enhancing the susceptibility of hypoxic tumor cells to chemotherapy.
The combination of a sensitizer with either radiation or a conventional anticancer drug helps overcome the problem of hypoxic cell resistance to cancer therapy. Chemosensitization in vitro usually is demonstrated by pretreating cells with a sensitizer under hypoxic conditions before exposure to the chemotherapeutic drug, often an alkylating agent, under aerobic conditions. This "preincubation effect" is attributed predominantly to a reduction of the sensitizer which occurs under hypoxic conditions.
Investigators have searched for improved hypoxic cell sensitizers that are non-toxic to aerobic and that concentrate more effectively in tumors. One hypoxic cell radiosensitizer is misonidazole (MISO), an electron-affinic 2-nitroimidazole which has shown some benefit in certain situations. However, MISO exhibits significant neurotoxicity and, consequently, the total dose of MISO that can be administered to a patient is limited. Another hypoxic cell sensitizer is etanidazole (SR-2508), a neutral compound which is more hydrophilic than MISO, is less neurotoxic, and can be administered to humans at about a threefold higher dose than MISO.
A third hypoxic cell sensitizer is pimondazole (Ro 03-8799), which contains a basic piperidine moiety and has a total dose limitation similar to MISO. A fourth compound, RSU-1069, is a bifunctional molecule containing a 2-nitroimidazole group and an alkylating aziridine. In experimental systems, RSU-1069 has a substantially greater activity than MISO, and is toxic to hypoxic cells in vitro at about a 100-fold lower concentration compared to the toxic concentration for aerobic cells.
Bioreductive radiosensitizers also have an ability to significantly enhance the activity of several chemotherapeutic agents, such as, e.g., cyclophosphamide, nitrosoureas, L-phenylalanine mustard (i.e., L-PAM or 4-[bis(2-chloroethyl)amino]-L-phenylalanine), cis-diamminedichloroplatinum(II) (i.e., cis-DDP) and doxorubicin, in vitro and in vivo. This enhancement of chemotherapeutic activity is known as chemosensitization or chemopotentiation.
Although significant progress has been made in developing bioreductive drugs as radio- and chemosensitizers, further development is necessary because none of the bioreductive drugs tested to date has shown impressive clinical results. Currently, therefore, there is a strong interest in targeting bioreductive agents to DNA in order to improve the radio- and chemosensitizing properties of such agents. Efforts to increase cytotoxic efficacy have centered on increasing the concentration of sensitizer within DNA as opposed to increasing the average intracellular concentration. Some investigators targeted DNA by combining an alkylating agent with bioreductive functional groups within the same drug (e.g., RSU-1069). Other investigators used transition metal coordination complexes such as platinum and ruthenium to target nitroaromatic radiosensitizers to DNA. However, these metal coordination complexes often are less effective as radiosensitizers than the free radiosensitizer molecule, even though the one electron reduction potential (a property related to sensitization efficiency) can be increased in some platinum complexes compared to the free sensitizer molecule.
Another approach to improve targeting of bioreductive agents to DNA involves using an intercalating moiety such as a phenanthridine or an acridine, which inserts itself between two adjacent sets of base pairs of the DNA. Non-covalent binding to DNA, such as through intercalation, permits migration of the radiosensitizer to DNA sites where radiation induced radicals are created.
For example, NLP-1, 5-[3-(2-nitro-1-imidazolyl)-propyl]phenanthridinium bromide, a 2-nitroimidazole-linked phenanthridine, has been synthesized. The synthesis, hypoxic cell cytotoxicity and radiosensitization of NLP-1 has been reported by R. Panicucci et al., Int. J. Radiat. Oncol. Biol. Phys., 16, pages 1039-1043 (1989), incorporated herein by reference. ##STR2##
An acridine-based hypoxia selective cytotoxin is preferred over the phenanthridine-based compound because acridine is a better intercalator than phenanthridine. Nitracrine, 1-nitro-acridine, is a potent hypoxia selective cytotoxin and a radiation sensitizer in mammalian cell cultures; however, rapid metabolism limits the radiosensitization efficacy of nitracrine in vivo. See, P. B. Roberts et al., Radiation Research, 123, pages 153-164 (1990), incorporated herein by reference.
An acridine-based hypoxia selective cytotoxin that is relatively stable in vivo therefore is preferred. In addition, it is preferred that the hypoxia selective cytotoxin does not bind tightly to DNA. For example, 1-nitracrine, which exhibits faster dissociation kinetics from DNA than the other nitroacridine isomers, is twenty times more potent as a sensitizer than other nitracrine isomers tested.
Papadopoulou-Rosenzweig et al. U.S. Pat. No. 5,294,715, incorporated herein by reference, discloses hypoxia selective cytotoxins having the structure ##STR3## wherein n is from 1 to 5, and NO.sub.2 is in at least one of the 2, 4 or 5-positions of the imidazole ring.
The compounds disclosed in Papadopoulou-Rosenzweig et al. U.S. Pat. No. 5,294,715 include an aromatic acridine moiety and are useful as radiosensitizers and chemosensitizers. These acridine-based compounds are electron affinic and exhibit strong DNA intercalating properties. However, the acridine-based compounds demonstrated a less than expected radiosensitization in vivo. This unexpectedly low radiosensitization has been attributed to restricted mobility of the acridine-based compounds along the DNA backbone, and to low extravascular diffusion in tumors. Mobility along the DNA backbone is considered a significant factor with respect to trapping radiation-induced radicals and providing good radiosensitization and chemosensitization efficacy.
Investigators therefore have continued efforts to develop a hypoxia selective cytotoxin and sensitizer having a lower affinity to bind to DNA and having enhanced efficacy. Accordingly, the present invention is directed to bioreductive cytotoxins which enhance the cytotoxic activities of ionizing radiation and chemotherapeutic agents to hypoxic cells, which are inherently cytotoxic to hypoxic cells, and which are essentially nontoxic to aerobic cells.